Rosemary B. Wright

Chromosome mapping of biological pathways by fluorescence-activated cell sorting and cell fusion: human interferon gamma receptor as a model system

Human chromosome 6 encodes both the interferon gamma receptor as well as the class I major histocompatability complex antigens, HLA-A, -B, and -C. However, the presence of chromosome 6 in somatic cell hybrids is insufficient to confer sensitivity to human interferon gamma (Hu-IFN-γ) as assayed by class I HLA induction; it is necessary for both human chromosomes 6 and 21 to reside in the hybrid to generate a response to Hu-IFN-γ. Treatment of such a hamster-human hybrid, Q72-18, with Hu-IFN-γ induces the class I HLA antigens. Q72-18 cells selected by fluorescence-activated cell sorting for the loss of class I HLA induction also lost human chromosome 21. Fusions of such cells to a hybrid that contains only human chromosome 21 reconstitutes HLA antigen induction by Hu-IFN-γ. Furthermore, fusions of hybrids containing a translocated human chromosome 6q and the HLA-B7 gene to a line containing only human chromosome 21 or a translocated 21q also reconstitutes HLA-B7 mRNA and antigen induction by Hu-IFN-γ. Thus the segregation of cells on the basis of a biological effect by fluorescence-activated cell sorting and reconstitution by hybrid fusion provides a strategy by which some biological pathways can be mapped at a chromosomal level.

Hydrocortisone binding receptor in the rat pituitary GH3 cell line

Abstract A receptor with specificity and high affinity for hydrocortisone (HC) has been found in the cytosol of GH 3 cells, a growth hormone (GH) producing culture. Scatchard analysis indicated that the interaction of [ 3 H]HC with the receptor has an apparent dissociation constant (K d ) of about 6.0 × 10 −9 M and a concentration of binding sites of approx. 1 × 10 −13 mol/mg cytosol protein. The second order association rate constant was determined to be 1.5 × 10 6 M −1 min −1 . The receptor activity is stable at 2°C for several hours, but is destroyed completely by heating at 37°C for 1 hour, or by treatment with pronase, only partially by RNase, but not by DNase. The binding of [ 3 H]HC to the cytosol receptor is inhibited by unlabeled progesterone (PR) or dexamethasone to the same extent as the inhibition by unlabeled HC. However, it is only partially inhibited by testosterone, 17-methyl-testosterone, 17α and 17β-estradiol, and 4-pregnen-20β-ol-3-one, and is unaffected by 5α-pregnan-3β,20β-diol. The biological role for these receptors in the regulation of GH synthesis is supported by the observations that the HC-stimulated production of GH is antagonized by PR, which competes with the binding of HC to the receptor.